Species: Mouse

Cell type: Melanoma

Transgenes: Enhanced green fluorescent protein (eGFP) with puromycin resistance (Puro) for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 1ug/mL puromycin

Description: B16F10-eGFP-Puro is a polyclonal population of the murine melanoma cell line B16F10 (ATCC^® CRL-6475^™) transduced with LV-eGFP-PGK-Puro (LV031) encoding enhanced green fluorescent protein (eGFP) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under control of the PGK promoter.

The lentiviral vector used is a self-inactivating (SIN) vector in which the viral enhancer and promoter has been deleted. Transcription inactivation of the LTR in the SIN provirus increases biosafety by preventing mobilization by replication competent viruses and enables regulated expression of the genes from the internal promoters without cis-acting effects of the LTR (Miyoshi et al., J Virol. 1998).

Cell Line Authentication: The parental B16F10 cell line was authenticated and are certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 27 STR loci.

Recommended uses:

In vitro: This is a high eGFP expressing cell line suitable for use as a positive control cell line in fluorescence assays to verify GFP expression in your lentiviral transduced cells.

In vivo: B16F10 cells form metastases in the lungs of mice. The in vivo growth of these metastases can be monitored using optical imaging for GFP fluorescence.

Note: In-life imaging for GFP fluorescence is not recommended due to high background autofluorescence. Tissues may be harvested post mortem for analysis by conventional fluorescence microscopy. For in vivo imaging, please use luciferase or the nonimmunogenic murine NIS reporter gene.

References on NIS imaging:

1. Fruthwirth et al. A whole body dual modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment responses in different. microenvironments. J. Nucl. Med 2014. 55(4): 686-94.

2. Penheiter et al. The sodium iodide symporter (NIS) as an imaging reporter for gene, viral and cell-based therapies. Curr Gene Ther. 2012, 12(1):33-47.

Morphology: Low- and high-density cell morphology (200x)

Transgene Validation:

Flow Cytometry for eGFP: B16F10-GFP-Puro (green) and control B16F10 (grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

Publications that used associated reagents: 

LV-eGFP-PGK-Puro (LV031): Shen et al. Immunovirotherapy with vesicular stomatitis virus and PD-L1 blockade enhances therapeutic outcome in murine acute myeloid leukemia. Blood. 2016. March 17: 127(11): 1449-58.